Multivalent pneumococcal polysaccharide-protein conjugate composition

ABSTRACT

An immunogenic composition having 13 distinct polysaccharide-protein conjugates and optionally, an aluminum-based adjuvant, is described. Each conjugate contains a capsular polysaccharide prepared from a different serotype of  Streptococcus pneumoniae  (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) conjugated to a carrier protein. The immunogenic composition, formulated as a vaccine, increases coverage against pneumococcal disease in infants and young children globally, and provides coverage for serotypes 6A and 19A that is not dependent on the limitations of serogroup cross-protection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/644,207, filed Dec. 22, 2006, which is a continuation-in-part of U.S.application Ser. No. 11/395,593, filed Mar. 31, 2006, which claims thebenefit of U.S. Provisional Application No. 60/669,605, filed Apr. 8,2005, each of which is hereby incorporated in its entirety by referenceherein.

FIELD OF THE INVENTION

The present invention relates generally to the field of medicine, andspecifically to microbiology, immunology, vaccines and the prevention ofinfection by a bacterial pathogen by immunization.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is a leading cause of meningitis, pneumonia,and severe invasive disease in infants and young children throughout theworld. The multivalent pneumococcal polysaccharide vaccines have beenlicensed for many years and have proved valuable in preventingpneumococcal disease in elderly adults and high-risk patients. However,infants and young children respond poorly to most pneumococcalpolysaccharides. The 7-valent pneumococcal conjugate vaccine (7vPnC,Prevnar®) was the first of its kind demonstrated to be highlyimmunogenic and effective against invasive disease and otitis media ininfants and young children. This vaccine is now approved in manycountries around the world. Prevnar contains the capsularpolysaccharides from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, eachconjugated to a carrier protein designated CRM₁₉₇. Prevnar coversapproximately 80-90%, 60-80%, and 40-80% of invasive pneumococcaldisease (IPD) in the US, Europe, and other regions of the world,respectively [1,2]. Surveillance data gathered in the years followingPrevnar's introduction has clearly demonstrated a reduction of invasivepneumococcal disease in US infants as expected (FIG. 1) [3,4].

Surveillance of IPD conducted in US infants prior to the introduction ofPrevnar demonstrated that a significant portion of disease due toserogroups 6 and 19 was due to the 6A (approximately one-third) and 19A(approximately one-fourth) serotypes [5,6]. Pneumococcal invasivedisease surveillance conducted in the US after licensure of Prevnarsuggests that a large burden of disease is still attributable toserotypes 6A and 19A (FIG. 1) [3]. Moreover, these two serotypes accountfor more cases of invasive disease than serotypes 1, 3, 5, and 7Fcombined (8.2 vs. 3.3 cases/100,000 children 2 years and under). Inaddition, serotypes 6A and 19A are associated with high rates ofantibiotic resistance (FIG. 2) [7,8,9]. While it is possible thatserogroup cross-protection will result in a decline of serotype 6A and19A disease as more children are immunized, there is evidence to suggestthat there will be a limit to the decline, and a significant burden ofdisease due to these serotypes will remain (see below).

Given the relative burden and importance of invasive pneumococcaldisease due to serotypes 1, 3, 5, 6A, 7F, and 19A, adding theseserotypes to the Prevnar formulation would increase coverage forinvasive disease to >90% in the US and Europe, and as high as 70%-80% inAsia and Latin America. This vaccine would significantly expand coveragebeyond that of Prevnar, and provide coverage for 6A and 19A that is notdependent on the limitations of serogroup cross-protection.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides generally a multivalentimmunogenic composition comprising 13 distinct polysaccharide-proteinconjugates, wherein each of the conjugates contains a capsularpolysaccharide from a different serotype of Streptococcus pneumoniaeconjugated to a carrier protein, together with a physiologicallyacceptable vehicle. Optionally, an adjuvant, such as an aluminum-basedadjuvant, is included in the formulation. More specifically, the presentinvention provides a 13-valent pneumococcal conjugate (13vPnC)composition comprising the seven serotypes in the 7vPnC vaccine (4, 6B,9V, 14, 18C, 19F and 23F) plus six additional serotypes (1, 3, 5, 6A, 7Fand 19A).

The present invention also provides a multivalent immunogeniccomposition, wherein the capsular polysaccharides are from serotypes 1,3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of Streptococcuspneumoniae and the carrier protein is CRM₁₉₇.

The present invention further provides a multivalent immunogeniccomposition,

wherein the capsular polysaccharides are from serotypes 1, 3, 4, 5, 6A,6B, 7F, 9v, 14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae, thecarrier protein is CRM₁₉₇, and the adjuvant is an aluminum-basedadjuvant, such as aluminum phosphate, aluminum sulfate and aluminumhydroxide. In a particular embodiment of the invention, the adjuvant isaluminum phosphate.

The present invention also provides a multivalent immunogeniccomposition, comprising polysaccharide-protein conjugates together witha physiologically acceptable vehicle, wherein each of the conjugatescomprises a capsular polysaccharide from a different serotype ofStreptococcus pneumoniae conjugated to a carrier protein, and thecapsular polysaccharides are prepared from serotype 3 and at least oneadditional serotype.

In one embodiment of this multivalent immunogenic composition, theadditional serotype is selected from the group consisting of serotypes1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In anotherembodiment, the carrier protein is CRM₁₉₇. In yet another embodiment,the composition comprises an adjuvant, such as an aluminum-basedadjuvant selected from aluminum phosphate, aluminum sulfate and aluminumhydroxide. In a particular embodiment, the adjuvant is aluminumphosphate.

The present invention also provides a multivalent immunogeniccomposition, comprising polysaccharide-protein conjugates together witha physiologically acceptable vehicle, wherein each of the conjugatescomprises a capsular polysaccharide from a different serotype ofStreptococcus pneumoniae conjugated to a carrier protein, and thecapsular polysaccharides are prepared from serotypes 4, 6B, 9V, 14, 18C,19F, 23F and at least one additional serotype.

In one embodiment of this multivalent immunogenic composition, theadditional serotype is selected from the group consisting of serotypes1, 3, 5, 6A, 7F, and 19A. In another embodiment, the carrier protein isCRM₁₉₇. In yet another embodiment, the composition comprises anadjuvant, such as an aluminum-based adjuvant selected from aluminumphosphate, aluminum sulfate and aluminum hydroxide. In a particularembodiment, the adjuvant is aluminum phosphate.

The present invention also provides a method of inducing an immuneresponse to a Streptococcus pneumoniae capsular polysaccharideconjugate, comprising administering to a human an immunologicallyeffective amount of any of the immunogenic compositions just described.

The present invention further provides that any of the immunogeniccompositions administered is a single 0.5 mL dose formulated to contain:2 μg of each saccharide, except for 6B at 4 μg; approximately 29 μgCRM₁₉₇ carrier protein; 0.125 mg of elemental aluminum (0.5 mg aluminumphosphate) adjuvant; and sodium chloride and sodium succinate buffer asexcipients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the changes in IPD rates by serotype in US children <2years of age from baseline (1998/1999) to 2001.

FIG. 2 depicts the distribution of pneumococcal isolates with resistanceto penicillin (PCN) in children <5 years of age (1998).

FIG. 3 depicts the reverse cumulative distribution curves (RCDC) of OPApost-third dose results from the D118-P16 Prevnar trial.

DETAILED DESCRIPTION OF THE INVENTION Inclusion of Prevnar Serotypes 4,6B, 9V, 14, 18C, 19F, 23F

Data from IPD surveillance between 1995-1998 estimated that the sevenserotypes in Prevnar were responsible for around 82% of IPD in children<2 years of age [5]. In Northern California, the site of the efficacytrial, the Prevnar serotypes accounted for 90% of all cases of IPD ininfants and young children [10]. Since introduction of the Prevnarvaccine in 2000, there has been a significant decrease in the overallIPD rates due to a decrease in disease due to the vaccine serotypes[3,4]. Therefore, there is no justification at this time to remove anyof the Prevnar serotypes from the next generation of pneumococcalconjugate vaccines but rather to add serotypes to obtain wider coverage.

Inclusion of Serotypes 1, 3, 5 and 7F

In the US, the rate of IPD caused by serotype 1 in children under theage of 5 years is <2%, about the same as for each of types 3 and 7F[1,6]. Serotypes 1 and 5 account for higher rates of IPD in USpopulations at high risk for invasive pneumococcal disease.Specifically, serotype 1 causes 3.5% of IPD in Alaskan native children<2 years of age, and 18% in children 2-4 years of age [11]. Bothserotype 1 and serotype 5 significantly cause disease in other parts ofthe world and in indigenous populations in developed countries[12,13,14].

Serotype 1 may also be associated with more severe disease as comparedwith other pneumococcal serotypes [15]. This observation is based on thedifference in rates of case identification between the US and Europe,and the associated difference in medical practice. Overall, theincidence of IPD is lower in Europe than in the US. However, the percentof IPD caused by serotype 1 in Europe is disproportionately higher thanin the US (6-7%, vs. 1-2%, respectively). In Europe, blood cultures areobtained predominantly from hospitalized children. In the US, it isroutine medical practice to obtain blood cultures in an outpatientsetting from children presenting with fever ≧39° C. and elevated whiteblood cell counts. Given the difference in medical practice, it ispostulated that the lower percent of disease caused by serotype 1 in theUS may be diluted by higher rates of other serotypes causing milderdisease, while the higher percent in Europe reflects more seriousdisease. In addition, seroepidemiology studies of children withcomplicated pneumonia demonstrate that serotype 1 is disproportionatelyrepresented [16,17,18]. This suggests that inclusion of serotype 1 mayreduce the amount of severe pneumococcal disease, as well as, contributeto a total reduction in invasive pneumococcal disease.

The addition of serotypes 3 and 7F will increase coverage against IPD inmost areas of the world by approximately 3%-7%, and in Asia by around9%. Thus, an 11-valent vaccine would cover 50% in Asia and around 80% ofIPD in all other regions [1,2]. These serotypes are also important withrespect to otitis media coverage [19]. In a multinational study ofpneumococcal serotypes causing otitis media, Hausdorff et al foundserotype 3 to be the 8th most common middle ear fluid isolate overall[20]. Serotype 3 accounted for up to 8.7% of pneumococcal serotypesassociated with otitis media. Thus, the importance of types 3 and 7F inotitis media, as well as in IPD, warrants their inclusion in apneumococcal conjugate vaccine.

However, attempts to produce a multivalent pneumococcal conjugatevaccine that exhibits significant immunogenicity with respect toserotype 3 polysaccharides have been unsuccessful. For example, in astudy of the immunogenicity and safety of an 11-valent pneumococcalprotein D conjugate vaccine (11-Pn-PD), no priming effect was observedfor serotype 3 in infants who had received three doses of the vaccinefollowed by a booster dose of either the same vaccine or a pneumococcalpolysaccharide vaccine (Nurkka et al., (2004) Ped. Inf. Dis. J.,23:1008-1014). In another study, opsonophagocytic assay (OPA) resultsfrom infants who had received doses of 11-Pn-PD failed to show antibodyresponses for serotype 3 at levels comparable to other tested serotypes(Gatchalian et al., 17^(th) Annual Meeting of the Eur. Soc. Paed. Inf.Dis. (ESPID), Poster No. 4, P1A Poster Session 1, Istanbul Turkey, Mar.27, 2001). In yet another study, which assessed the efficacy of an11-Pn-PD in the prevention of acute otitis media, the vaccine did notprovide protection against episodes caused by serotype 3 (Prymula et al.www.thelancet.com, Vol. 367: 740-748 (Mar. 4, 2006)). Accordingly, apneumococcal conjugate vaccine comprising capsular polysaccharides fromserotype 3 and capable of eliciting an immunogenic response to serotype3 polysaccharides provides a significant improvement over the existingstate of the art.

Inclusion of Serotypes 6A and 19A

a. Epidemiology of Serotypes 6A and 19A

Surveillance data in the literature suggest that serotypes 6A and 19Aaccount for more invasive pneumococcal disease in US children <2 yearsof age than serotypes 1, 3, 5, and 7F combined (FIG. 1) [1,5]. Inaddition, these serotypes are commonly associated with antibioticresistance (FIG. 2) and play an important role in otitis media[6,19,20].The ability of the current Prevnar vaccine to protect against diseasedue to 6A and 19A is not clear. The rationale for inclusion of 6A and19A components in a 13vPnC vaccine is discussed below.

b. Responses to 6A and 19A Induced by 6B and 19F Polysaccharides

The licensed unconjugated pneumococcal polysaccharide vaccines (for usein persons at least two years of age) have contained 6A or 6B capsularpolysaccharide but not both [21]. Immunogenicity data generated at thetime of formulation of the 23-valent pneumococcal polysaccharide vaccinedemonstrated that a 6B monovalent vaccine induced antibody to both the6A and 6B capsules. The data from several trials assessing IgG andopsonophagocytic assay (OPA) responses in a variety of populations withfree polysaccharide and with pneumococcal conjugate vaccines suggestedthat IgG responses to 6A are induced by 6B antigens, but the responsesare generally lower, and the OPA activity with 6A organisms is differentthan with 6B organisms [22,23,24,25]. In addition, subjects respondingwith high 6B antibody may have little or no activity against 6A.

In contrast to the chemical composition of the 6A and 6B capsularpolysaccharides where there exists a high degree of similarity, the 19Aand 19F capsules are quite different due to the presence of twoadditional side chains in the 19A polysaccharide. Not surprisingly,immune responses measured in human volunteers immunized with 19Fpolysaccharide vaccine showed that responses to 19F were induced in 80%of subjects, but only 20% of subjects had a response to 19A [26]. Lowlevels of cross-reactive IgG and OPA responses to serotype 19A afterimmunization with 19F polysaccharide have also been documented in trialswith conjugate vaccines as well [24,26].

Internal data on cross-reactive OPA responses to 6A and 19A have beengenerated from the 7vPnC bridging trial (D118-P16) conducted in USinfants (FIG. 3). These studies are consistent with the findings ofothers, and demonstrate induction of cross-reactive functional antibodyto 6A polysaccharide after immunization with 6B polysaccharide, althoughat a lower level, and very little functional antibody to 19A afterimmunization with 19F.

Impact of 6B and 19F Immunization on 6A and 19A in Animal Models

Animal models have been used to evaluate the potential forcross-protection with polysaccharide immunization. In an otitis mediamodel developed by Giebink et al., chinchillas were immunized with atetravalent polysaccharide outer membrane protein (OMP) conjugatevaccine (containing 6B, 14, 19F, 23F saccharides) or placebo [27]. Inthis trial there appeared to be some cross-protection for 6A; howeverthis did not reach statistical significance and the level of protectionwas lower than with 6B against otitis media. In this same model therewas 100% protection against 19F otitis media, but only 17% protectionagainst 19A otitis media.

Saeland et al. used sera from infants immunized with an 8-valentpneumococcal tetanus conjugate vaccine (containing 6B and 19F) topassively immunize mice prior to an intranasal challenge with 6Aorganisms, in a lung infection model [28]. Of the 59 serum samples, 53%protected mice against bacteremia with 6B and 37% protected against 6A.Mice passively immunized with sera from infants immunized with fourdoses of an 11-valent pneumococcal conjugate vaccine (containing 19Fconjugated to tetanus toxoid) were given an intranasal challenge with19A organisms in the same model [29]. Of 100 mice passively immunizedand then challenged, 60 mice had no 19A organisms detected in lungtissue, whereas organisms were identified in all mice given salineplacebo. However, passive immunization did not protect against challengewith 19F organisms in this model; therefore, the relevance of the modelfor serogroup 19 is questionable. In general these models provideevidence of some biological impact of 6B immunization on 6A organismsalthough the effect on the heterologous serotype was not as great asthat observed with the homologous serotype. The impact of 19Fimmunization on 19A organisms is not well understood from these models.

Impact of 6B and 19F Polysaccharide Conjugate Immunization on 6A and 19ADisease in Efficacy/Effectiveness Trials

The number of cases of disease due to the 6B, 6A, 19F and 19A serotypesin 7vPnC and 9vPnC (7vPnC plus serotypes 1 and 5) efficacy trials isnoted in Table 1 [30,10,31]. The numbers of invasive disease cases aretoo small to allow any conclusions to be drawn for serotypes 6A and 19A.However, the Finnish otitis media trial generated a large number ofpneumococcal isolates [32]. In the per protocol analysis 7vPnC was 84%(95% Cl 62%, 93%) efficacious against otitis media due to serotype 6Band 57% (95% Cl 24%, 76%) efficacious against otitis media due toserotype 6A (Table 1). In contrast, serotype-specific efficacy with the7vPnC was not demonstrated for otitis media due to either 19F or 19A.

TABLE 1 Cases of Pneumococcal Disease Due to Serotypes 6B, 6A, 19F, and19A in Efficacy Trials with the 7vPnC and 9vPnC Vaccines 6B 6A 19F 19APnC Contr. PnC Contr. PnC Contr. PnC Contr. Kaiser Efficacy Trial -7vPnC 1 7 0 1  2* 13 0 1 (ITT) Navajo Efficacy Trial - 7vPnC 0 5 1 0 1 11 0 (ITT) South African Efficacy Trial - 1 2 1 0 0 1 3 1 9vPnC HIV (−)(ITT) South African Efficacy Trial - 1 7 3 10 2 3 2 3 9vPnC HIV (+)(ITT) Finnish Otitis Media Trial -  9* 56 19* 45 43  58 17 26 7vPnC (PP)*Statistically significant efficacy demonstrated From references 30, 10and 33, and personal communications Contr = control ITT = intention totreat analysis PP = per protocol analysis

Post-marketing IPD surveillance data is also available from acase-control trial conducted by the Centers for Disease Control toevaluate the effectiveness of Prevnar [33]. Cases of pneumococcalinvasive disease occurring in children 3 to 23 months of age wereidentified in the surveillance laboratories and matched with threecontrol cases by age and zip code. After obtaining consent, medical andimmunization history (subjects were considered immunized if they hadreceived at least one dose of Prevnar) was obtained from parents andmedical providers for cases and controls. The preliminary results werepresented at the 2003 ICAAC meeting and a summary of the findings for6B, 19F, 19A and 6A disease is presented in Table 2. These data indicatethat Prevnar is able to prevent disease due to 6A, although at a levelthat may be somewhat lower than serotype 6B disease. These data alsoindicate that the cross-protection for invasive disease due to 19A islimited.

TABLE 2 Preliminary results of a Case Control Trial Performed by the CDC(presented at ICAAC, 2003) VE* Serotype Informative Sets, n (95% Cl)Vaccine Type, All 115 94 (87, 97) Vaccine Related, All 36 70 (38, 86)Non-Vaccine Type, All 43 −4 (−106, 48)   6B 27 94 (72, 99) 19F 19 73(16, 92) 6A 15 87 (53, 97) 19A 16 40 (−87, 80)  *Vaccine effectivenesscomparing vaccinated (≧1 dose) vs. unvaccinated, and adjusted forunderlying conditions Reference 40 and personal/confidentialcommunication

A published analysis [3] of the use of Prevnar also indicated thatserotypes 6B and 19F conferred a moderate reduction in IPD caused byserotypes 6A and 19A among children under two years of age (Table 1 in[3]). Disease rates among unimmunized adults caused by serotypes 6A, 9A,9L, 9N, 18A, 18B, 18F, 19A, 19B, 19C, 23A and 23B (“all vaccine-relatedserotypes”) were somewhat reduced (Table 2 in [3]). These data establishthat herd immunity from the use of Prevnar in children under two yearsof age was modest for serotypes 6A and 19A, and provide a basis for theinclusion of serotypes 6A and 19A in the 13vPnC vaccine of thisinvention.

Conclusion for Addition of 6A and 19A

The post-marketing surveillance data and the case-control study resultsnoted in FIG. 1 and Table 2 with the 7vPnC vaccine suggest that,consistent with the other information on immune responses andperformance in the animals models described above, there may be somecross-protection against 6A disease, but to a lesser extent than to 6Bdisease. Furthermore, it appears the protection against 19A is limited.Therefore, a 13vPnC vaccine containing serotypes 6A and 19A providescoverage that is not dependent on the limitations of serogroupcross-protection by serotypes 6B and 19F.

Accordingly, the present invention provides a multivalent immunogeniccomposition comprising 13 distinct polysaccharide-protein conjugates,wherein each of the conjugates contains a different capsularpolysaccharide conjugated to a carrier protein, and wherein the capsularpolysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae, together with aphysiologically acceptable vehicle. One such carrier protein is thediphtheria toxoid designated CRM₁₉₇. The immunogenic composition mayfurther comprise an adjuvant, such as an aluminum-based adjuvant, suchas aluminum phosphate, aluminum sulfate and aluminum hydroxide.

Capsular polysaccharides are prepared by standard techniques known tothose skilled in the art. In the present invention, capsularpolysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V,14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae. Thesepneumococcal conjugates are prepared by separate processes andformulated into a single dosage formulation. For example, in oneembodiment, each pneumococcal polysaccharide serotype is grown in asoy-based medium. The individual polysaccharides are then purifiedthrough centrifugation, precipitation, ultra-filtration, and columnchromatography. The purified polysaccharides are chemically activated tomake the saccharides capable of reacting with the carrier protein.

Once activated, each capsular polysaccharide is separately conjugated toa carrier protein to form a glycoconjugate. In one embodiment, eachcapsular polysaccharide is conjugated to the same carrier protein. Inthis embodiment, the conjugation is effected by reductive amination.

The chemical activation of the polysaccharides and subsequentconjugation to the carrier protein are achieved by conventional means.See, for example, U.S. Pat. Nos. 4,673,574 and 4,902,506 [34,35].

Carrier proteins are preferably proteins that are non-toxic andnon-reactogenic and obtainable in sufficient amount and purity. Carrierproteins should be amenable to standard conjugation procedures. In aparticular embodiment of the present invention, CRM₁₉₇ is used as thecarrier protein.

CRM₁₉₇ (Wyeth, Sanford, N.C.) is a non-toxic variant (i.e., toxoid) ofdiphtheria toxin isolated from cultures of Corynebacterium diphtheriastrain C7 (β197) grown in casamino acids and yeast extract-based medium.CRM₁₉₇ is purified through ultra-filtration, ammonium sulfateprecipitation, and ion-exchange chromatography. Alternatively, CRM₁₉₇ isprepared recombinantly in accordance with U.S. Pat. No. 5,614,382, whichis hereby incorporated by reference. Other diphtheria toxoids are alsosuitable for use as carrier proteins.

Other suitable carrier proteins include inactivated bacterial toxinssuch as tetanus toxoid, pertussis toxoid, cholera toxoid (e.g., asdescribed in International Patent Application WO2004/083251 [38]), E.coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa.Bacterial outer membrane proteins such as outer membrane complex c(OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcalsurface protein A (PspA), pneumococcal adhesin protein (PsaA), C5apeptidase from Group A or Group B streptococcus, or Haemophilusinfluenzae protein D, can also be used. Other proteins, such asovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA)or purified protein derivative of tuberculin (PPD) can also be used ascarrier proteins.

After conjugation of the capsular polysaccharide to the carrier protein,the polysaccharide-protein conjugates are purified (enriched withrespect to the amount of polysaccharide-protein conjugate) by a varietyof techniques. These techniques include concentration/diafiltrationoperations, precipitation/elution, column chromatography, and depthfiltration. See examples below.

After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention, whichcan be used as a vaccine. Formulation of the immunogenic composition ofthe present invention can be accomplished using art-recognized methods.For instance, the 13 individual pneumococcal conjugates can beformulated with a physiologically acceptable vehicle to prepare thecomposition. Examples of such vehicles include, but are not limited to,water, buffered saline, polyols (e.g., glycerol, propylene glycol,liquid polyethylene glycol) and dextrose solutions.

In certain embodiments, the immunogenic composition will comprise one ormore adjuvants. As defined herein, an “adjuvant” is a substance thatserves to enhance the immunogenicity of an immunogenic composition ofthis invention. Thus, adjuvants are often given to boost the immuneresponse and are well known to the skilled artisan. Suitable adjuvantsto enhance effectiveness of the composition include, but are not limitedto:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example,

-   -   (a) MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene,        0.5% Tween 80, and 0.5% Span 85 (optionally containing various        amounts of MTP-PE (see below, although not required)) formulated        into submicron particles using a microfluidizer such as Model        110Y microfluidizer (Microfluidics, Newton, Mass.),    -   (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5%        pluronic-blocked polymer L121, and thr-MDP (see below) either        microfluidized into a submicron emulsion or vortexed to generate        a larger particle size emulsion, and    -   (c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, Mont.)        containing 2% Squalene, 0.2% Tween 80, and one or more bacterial        cell wall components from the group consisting of 3-O-deaylated        monophosphorylipid A (MPL™) described in U.S. Pat. No. 4,912,094        (Corixa), trehalose dimycolate (TDM), and cell wall skeleton        (CWS), preferably MPL+CWS (Detox™);

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (U.S. Pat. No. 5,057,540) may be used or particlesgenerated therefrom such as ISCOMs (immunostimulating complexes);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,which is also know as 529 (formerly known as RC529), which is formulatedas an aqueous form or as a stable emulsion, synthetic polynucleotidessuch as oligonucleotides containing CpG motif(s) (U.S. Pat. No.6,207,646);

(5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules B7-1 and B7-2, etc.;

(6) detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT) either in a wild-type or mutant form, for example,where the glutamic acid at amino acid position 29 is replaced by anotheramino acid, preferably a histidine, in accordance with publishedinternational patent application number WO 00/18434 (see also WO02/098368 and WO 02/098369), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129(see, e.g., WO 93/13302 and WO 92/19265); and

(7) other substances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

The vaccine formulations of the present invention can be used to protector treat a human susceptible to pneumococcal infection, by means ofadministering the vaccine via a systemic or mucosal route. Theseadministrations can include injection via the intramuscular,intraperitoneal, intradermal or subcutaneous routes; or via mucosaladministration to the oral/alimentary, respiratory or genitourinarytracts. In one embodiment, intranasal administration is used for thetreatment of pneumonia or otitis media (as nasopharyngeal carriage ofpneumococci can be more effectively prevented, thus attenuatinginfection at its earliest stage).

The amount of conjugate in each vaccine dose is selected as an amountthat induces an immunoprotective response without significant, adverseeffects. Such amount can vary depending upon the pneumococcal serotype.Generally, each dose will comprise 0.1 to 100 μg of polysaccharide,particularly 0.1 to 10 μg, and more particularly 1 to 5 μg.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. Following an initial vaccination, subjectscan receive one or several booster immunizations adequately spaced.

In a particular embodiment of the present invention, the 13vPnC vaccineis a sterile liquid formulation of pneumococcal capsular polysaccharidesof serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23Findividually conjugated to CRM₁₉₇. Each 0.5 mL dose is formulated tocontain: 2 μg of each saccharide, except for 6B at 4 μg; approximately29 μg CRM₁₉₇ carrier protein; 0.125 mg of elemental aluminum (0.5 mgaluminum phosphate) adjuvant; and sodium chloride and sodium succinatebuffer as excipients. The liquid is filled into single dose syringeswithout a preservative. After shaking, the vaccine is a homogeneous,white suspension ready for intramuscular administration.

The choice of dose level for the 13vPnC vaccine is similar to themarketed 7vPnC vaccine (Prevnar). The 2 μg saccharide dose level wasselected for all serotypes, except for 6B, which is at 4 μg per dose.The 7vPnC vaccine has shown desirable safety, immunogenicity, andefficacy against IPD in the 2 μg saccharide dose level for serotypes 4,9V, 14, 18C, 19F and 23F, and at the 4 μg dose for 6B.

The immunization schedule can follow that designated for the 7vPnCvaccine. For example, the routine schedule for infants and toddlersagainst invasive disease caused by S. pneumoniae due to the serotypesincluded in the 13vPnC vaccine is 2, 4, 6 and 12-15 months of age. Thecompositions of this invention are also suitable for use with olderchildren, adolescents and adults.

The compositions of this invention may further include one or moreadditional antigens for use against otitis media caused by infectionwith other bacteria. Such bacteria include nontypable Haemophilusinfluenza, Moraxella catarrhalis (formerly known as Branhamellacatarrhalis) and Alloiococcus otitidis.

Examples of nontypable Haemophilus influenzae antigens suitable forinclusion include the P4 protein, also known as protein “e” (U.S. Pat.No. 5,601,831; International Patent Application WO03/078453), the P6protein, also known as the PAL or the PBOMP-1 protein (U.S. Pat. No.5,110,908; International Patent Application WO0100790), the P5 protein(U.S. Reissue Pat. No. 37,741), the Haemophilus adhesion and penetrationprotein (U.S. Pat. Nos. 6,245,337 and 6,676,948), the LKP tip adhesinprotein (U.S. Pat. No. 5,643,725) and the NucA protein (U.S. Pat. No.6,221,365).

Examples of Moraxella catarrhalis antigens suitable for inclusioninclude the UspA2 protein (U.S. Pat. Nos. 5,552,146, 6,310,190), the CDprotein (U.S. Pat. No. 5,725,862), the E protein (U.S. Pat. No.5,948,412) and the 74 kilodalton outer membrane protein (U.S. Pat. No.6,899,885).

Examples of Alloiococcus otitidis antigens suitable for inclusioninclude those identified in International Patent ApplicationWO03/048304.

The compositions of this invention may also include one or more proteinsfrom Streptococcus pneumoniae. Examples of Streptococcus pneumoniaeproteins suitable for inclusion include those identified inInternational Patent Application WO02/083855, as well as that describedin International Patent Application WO02/053761.

The compositions of this invention may further include one or moreproteins from Neisseria meningitidis type B. Examples of Neisseriameningitidis type B proteins suitable for inclusion include thoseidentified in International Patent Applications WO03/063766,WO2004/094596, WO01/85772, WO02/16612 and WO01/87939.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theinvention.

EXAMPLES Example 1 Preparation of S. Pneumoniae Capsular PolysaccharideSerotype 1 Preparation of Master and Working Cell Banks

S. pneumoniae serotype 1 was obtained from the American Type CultureCollection, ATCC, strain 6301. Several generations of seed stocks werecreated in order to expand the strain and remove components of animalorigin (generations F1, F2, and F3). Two additional generations of seedstocks were produced. The first additional generation was made from anF3 vial, and the subsequent generation was made from a vial of the firstadditional generation. Seed vials were stored frozen (<−70° C.) withsynthetic glycerol as a cryopreservative. In addition to frozen vials,lyophilized vials were prepared for the F4 generation. For cell bankpreparation, all cultures were grown in a soy-based medium. Prior tofreezing, cells were concentrated by centrifugation, spent medium wasremoved, and cell pellets were re-suspended in fresh medium containing acryopreservative, such as synthetic glycerol.

Fermentation and Harvesting

Cultures from the working cell bank were used to inoculate seed bottlescontaining a soy-based medium. The bottles were incubated at 36° C.±2°C. without agitation until growth requirements were met. A seed bottlewas used to inoculate a seed fermentor containing soy-based medium. A pHof about 7.0 was maintained with sterile sodium carbonate solution.After the target optical density was reached, the seed fermentor wasused to inoculate the production fermentor containing soy-based medium.The pH was maintained with sterile sodium carbonate solution. Thefermentation was terminated after cessation of growth or when theworking volume of the fermentor was reached. An appropriate amount ofsterile 12% deoxycholate sodium was added to the culture to lyse thebacterial cells and release cell-associated polysaccharide. Afterlysing, the fermentor contents were cooled. The pH of the lysed culturebroth was adjusted to approximately pH 6.6 with acetic acid. The lysatewas clarified by continuous flow centrifugation followed by depthfiltration and 0.45 μm microfiltration.

In an alternate process, the fermentation pH of about 7.0 was maintainedwith 3N NaOH. After the target optical density was reached, the seedfermentor was used to inoculate the production fermentor containingsoy-based medium. The pH was maintained with 3N NaOH. The fermentationwas terminated after cessation of growth or when the working volume ofthe fermentor was reached. An appropriate amount of sterile 12%deoxycholate sodium was added to the culture to obtain a 0.12%concentration in the broth, to lyse the bacterial cells and releasecell-associated polysaccharide. After lysing, the fermentor contentswere held, with agitation, for a time interval between 8 and 24 hours ata temperature between 7° C. and 13° C., to assure that complete cellularlysis and polysaccharide release had occurred. Agitation during thishold period prevented lysate sediment from settling on the fermentorwalls and pH probe, thereby allowing the pH probe integrity to bemaintained. Next, the pH of the lysed culture broth was adjusted toapproximately pH 5.0 with 50% acetic acid. After a hold time withoutagitation, for a time interval between 12 and 24 hours at a temperaturebetween 15° C. and 25° C., a significant portion of the previouslysoluble proteins dropped out of solution as a solid precipitate withlittle loss or degradation of the polysaccharide, which remained insolution. The solution with the precipitate was then clarified bycontinuous flow centrifugation followed by depth filtration and 0.45 μmmicrofiltration.

Purification

The purification of the pneumococcal polysaccharide consisted of severalconcentration/diafiltration operations, precipitation/elution, columnchromatography, and depth filtration steps. All procedures wereperformed at room temperature unless otherwise specified.

Clarified broth from the fermentor cultures of S. pneumoniae serotype 1were concentrated and diafiltered using a 100 kDa MWCO (kilodaltonmolecular weight cutoff) filter. Diafiltration was accomplished usingsodium phosphate buffer at neutral pH. Diafiltration removed the lowmolecular weight medium components from the higher molecular weightbiopolymers such as nucleic acid, protein and polysaccharide.

The polysaccharide was precipitated from the concentrated anddiafiltered solution by adding hexadecyltrimethyl ammonium bromide (HB)from a stock solution to give a final concentration of 1% HB (w/v). Thepolysaccharide/HB precipitate was captured on a depth filter and thefiltrate was discarded. The polysaccharide precipitate was resolubilizedand eluted by recirculating a sodium chloride solution through theprecipitate-containing depth filter. The filters were then rinsed withadditional sodium chloride solution.

Sodium iodide (NaI) was added to the polysaccharide solution from astock NaI solution to achieve a final concentration of 0.5% toprecipitate HB. The precipitate was removed by depth filtration. Thefiltrate contains the target polysaccharide. The precipitation vesseland the filter were rinsed with a NaCl/NaI solution and the rinse wascombined with the partially purified polysaccharide solution. The filterwas discarded. The polysaccharide was then filtered through a 0.2 μmfilter.

The polysaccharide solution was concentrated on a 30 kDa MWCOultrafilter and diafiltered with a sodium chloride solution.

The partially purified polysaccharide solution was further purified byfiltration through a depth filter impregnated with activated carbon.After filtration, the carbon filter was rinsed with a sodium chloridesolution. The rinse is combined with the polysaccharide solution, whichis then filtered through a 0.2 μm filter.

The polysaccharide solution was concentrated on a 30 kDa MWCOultrafilter and adjusted with a 1M sodium phosphate buffer to achieve afinal concentration of 0.025 M sodium phosphate. The pH was checked andadjusted to 7.0±0.2.

The ceramic hydroxyapatite (HA) column was equilibrated with sodiumphosphate buffer containing sodium chloride to obtain the appropriateconductivity (<15 μS). The polysaccharide solution was then loaded ontothe column. Under these conditions, impurities bound to the resin andthe polysaccharide was recovered in the flow-through from the column.The polysaccharide solution was filtered through 0.2 μm inline filterslocated before and after the column.

The polysaccharide solution was concentrated using a 30 kDa MWCO filter.The concentrate was then diafiltered with Water for Injection (WFI).

The diafiltered polysaccharide solution was filtered through a 0.2 μmmembrane filter into polypropylene bottles. Samples were removed forrelease testing and the purified polysaccharide was stored frozen at−25°±5° C.

Characterization

The 1H-NMR data was consistent with the chemical structure by theassignment of signals assigned to the protons of the polysaccharidemolecule. The 1H-NMR spectrum showed a series of well-resolved signals(protons from the methyl group) for the quantitation of the O-acetylfunctional group in the polysaccharide.

The identity of the monovalent polysaccharide was confirmed bycountercurrent immunoelectrophoresis using specific antisera.

High performance gel filtration chromatography coupled with refractiveindex and multiangle laser light scattering (MALLS) detectors was usedin conjunction with the sample concentration to calculate the molecularweight.

Size exclusion chromatography media (CL-4B) was used to profile therelative molecular size distribution of the polysaccharide.

Example 2 Preparation of Serotype 1 Pneumococcal Saccharide CRM₁₉₇Conjugate Activation and Conjugation

Containers of purified polysaccharide were thawed and combined in areaction vessel. To the vessel, 0.2 M sodium carbonate, pH 9.0 was addedfor partial deacetylation (hydrolysis) for 3 hours at 50° C. Thereaction was cooled to 20° C. and neutralization was performed by 0.2 Macetic acid. Oxidation in the presence of sodium periodate was performedby incubation at 2-8° C., and the mixture was stirred for 15-21 hours.

The activation reaction mixture was concentrated and diafiltered 10×with 0.9% NaCl using a 30K MWCO membrane. The retentate was 0.2 μmfiltered. The activated saccharide was filled into 100 mL glasslyophilization bottles and shell-frozen at −75° C. and lyophilized.

“Shell-freezing” is a method for preparing samples for lyophilization(freeze-drying). Flasks are automatically rotated by motor drivenrollers in a refrigerated bath containing alcohol or any otherappropriate fluid. A thin coating of product is evenly frozen around theinside “shell” of a flask, permitting a greater volume of material to besafely processed during each freeze-drying run. These automatic,refrigerated units provide a simple and efficient means of pre-freezingmany flasks at a time, producing the desired coatings inside, andproviding sufficient surface area for efficient freeze-drying.

Bottles of lyophilized material were brought to room temperature andresuspended in CRM₁₉₇ solution at a saccharide/protein ratio of 2:1. Tothe saccharide/protein mixture 1M sodium phosphate buffer was added to afinal 0.2M ionic strength and a pH of 7.5, then sodium cyanoborohydridewas added. The reaction was incubated at 23° C. for 18 hours, followedby a second incubation at 37° C. for 72 hours. Following thecyanoborohydride incubations, the reaction mixture was diluted with coldsaline followed by the addition of 1M sodium carbonate to adjust thereaction mixture to pH 9.0. Unreacted aldehydes were quenched byaddition of sodium borohydride by incubation at 23° C. for 3-6 hours.

The reaction mixture was diluted 2-fold with saline and transferredthrough a 0.45-5 μm prefilter into a retentate vessel. The reactionmixture is diafiltered 30× with 0.15 M phosphate buffer, pH 6, and 20×with saline. The retentate was filtered through a 0.2 μm filter.

The conjugate solution was diluted to a target of 0.5 mg/mL in 0.9%saline, and then sterile filtered into final bulk concentrate (FBC)containers in a Class 100 hood. The conjugate was stored at 2-8° C.

Characterization

Size exclusion chromatography media (CL-4B) was used to profile therelative molecular size distribution of the conjugate.

The identity of the conjugate was confirmed by the slot-blot assay usingspecific antisera.

The saccharide and protein concentrations were determined by the uronicacid and Lowry assays, respectively. The ratio of saccharide to proteinin the covalently bonded conjugate complex was obtained by thecalculation:

${Ratio} = \frac{{µg}\text{/}{mL}\mspace{14mu} {saccharide}}{{µg}\text{/}{mL}\mspace{14mu} {protein}}$

O-acetyl content was measured by the Hestrin method (Hestrin et. al., J.Biol. Chem. 1949, 180, p. 249). The ratio of O-acetyl concentration tototal saccharide concentration gave μmoles of O-acetyl per mg ofsaccharide.

Example 3 Preparation of S. Pneumoniae Capsular Polysaccharide Serotype3 Preparation of Master and Working Cell Banks

S. pneumoniae serotype 3 was obtained from Dr. Robert Austrian,University of Pennsylvania, Philadelphia, Pa. For preparation of thecell bank system, see Example 1.

Fermentation and Harvesting

Cultures from the working cell bank were used to inoculate seed bottlescontaining soy-based medium. The bottles were incubated at 36° C.±2° C.without agitation until growth requirements were met. A seed bottle wasused to inoculate a seed fermentor containing soy-based medium. A pH ofabout 7.0 was maintained with sterile sodium carbonate solution. Afterthe target optical density was reached, the seed fermentor was used toinoculate an intermediate seed fermentor. After the target opticaldensity was reached, the intermediate seed fermentor was used toinoculate the production fermentor. The pH was maintained with sterilesodium carbonate solution. The fermentation was terminated after theworking volume of the fermentor was reached. An appropriate amount ofsterile 12% sodium deoxycholate was added to the culture to lyse thebacterial cells and release cell-associated polysaccharide. Afterlysing, the fermentor contents were cooled. The pH of the lysed culturebroth was adjusted to approximately pH 6.6 with acetic acid. The lysatewas clarified by continuous flow centrifugation followed by depthfiltration and 0.45 μm microfiltration.

Purification

The purification of the pneumococcal polysaccharide consisted of severalconcentration/diafiltration operations, precipitation/elution, columnchromatography, and depth filtration steps. All procedures wereperformed at room temperature unless otherwise specified.

Clarified broth from the fermentor cultures of S. pneumoniae serotype 3were concentrated and diafiltered using a 100 kDa MWCO filter.Diafiltration was accomplished using sodium phosphate buffer at neutralpH. Diafiltration removed the low molecular weight medium componentsfrom the higher molecular weight biopolymers such as nucleic acid,protein and polysaccharide.

Prior to the addition of hexadecyltrimethyl ammonium bromide (HB), acalculated volume of a NaCl stock solution was added to the concentratedand diafiltered polysaccharide solution to give a final concentration of0.25 M NaCl. The polysaccharide was then precipitated by adding HB froma stock solution to give a final concentration of 1% HB (w/v). Thepolysaccharide/HB precipitate was captured on a depth filter and thefiltrate was discarded. The polysaccharide precipitate was resolubilizedand eluted by recirculating a sodium chloride solution through theprecipitate-containing depth filter. The filters were then rinsed withadditional sodium chloride solution.

Sodium iodide (NaI) was added to the polysaccharide solution from astock NaI solution to achieve a final concentration of 0.5% toprecipitate HB. The precipitate was removed by depth filtration. Thefiltrate contained the target polysaccharide. The precipitation vesseland the filter were rinsed with a NaCl/NaI solution and the rinse wascombined with the partially purified polysaccharide solution. The filterwas discarded. The polysaccharide was then filtered through a 0.2 μmfilter.

The polysaccharide solution was concentrated on a 30 kDa MWCOultrafilter and diafiltered with a sodium chloride solution.

The partially purified polysaccharide solution was further purified byfiltration through a depth filter impregnated with activated carbon.After filtration, the carbon filter was rinsed with a sodium chloridesolution. The rinse was combined with the polysaccharide solution, whichwas then filtered through a 0.2 μm filter.

The polysaccharide solution was concentrated on a 30 kDa MWCOultrafilter and adjusted with a 1M sodium phosphate buffer to achieve afinal concentration of 0.025M sodium phosphate. The pH was checked andadjusted to 7.0±0.2.

The ceramic hydroxyapatite (HA) column was equilibrated with sodiumphosphate buffer containing sodium chloride to obtain the appropriateconductivity (15 μS). The polysaccharide solution was then loaded ontothe column. Under these conditions, impurities bound to the resin andthe polysaccharide was recovered in the flow-through from the column.The polysaccharide was flushed through the column with buffer and wasfiltered through a 0.2 μm filter.

The polysaccharide solution was concentrated using a 30 kDa MWCO filter.The concentrate was then diafiltered with WFI.

The diafiltered polysaccharide solution was filtered through a 0.2 μmmembrane filter into stainless steel containers. Samples were removedfor release testing and the purified polysaccharide was stored frozen at−25°+5° C.

Characterization

The ¹H-NMR data was consistent with the chemical structure by theassignment of signals assigned to the protons of the polysaccharidemolecule.

The identity of the monovalent polysaccharide was confirmed bycountercurrent immunoelectrophoresis using specific antisera.

High performance gel filtration chromatography, coupled with refractiveindex and multiangle laser light scattering (MALLS) detectors, was usedin conjunction with the sample concentration to calculate the molecularweight.

Size exclusion chromatography media (CL-4B) was used to profile therelative molecular size distribution of the polysaccharide.

Example 4 Preparation of Serotype 3 Pneumococcal Saccharide CRM₁₉₇Conjugate Activation and Conjugation

Containers of purified serotype 3 saccharide were thawed and combined ina reaction vessel. To the vessel, WFI and 2M acetic acid were added to afinal concentration of 0.2M and 2 mg/mL saccharide. The temperature ofthe solution was raised to 85° C. for one hour to hydrolyze thepolysaccharide. The reaction was cooled to ≦25° C. and 1M magnesiumchloride was added to a final concentration of 0.1M. Oxidation in thepresence of sodium periodate was performed by incubation for 16-24 hoursat 23° C.

The activation reaction mixture was concentrated and diafiltered 10×with WFI using a 100K MWCO membrane. The retentate was filtered througha 0.2-μm filter.

For compounding, 0.2M sodium phosphate, pH 7.0, was added to theactivated saccharide to a final concentration of 10 mM and a pH of6.0-6.5. CRM₁₉₇ carrier protein was mixed with the saccharide solutionto a ratio of 2 g of saccharide per 1 g of CRM₁₉₇. The combinedsaccharide/protein solution was filled into 100 mL glass lyophilizationbottles with a 50 mL target fill, shell-frozen at −75° C., andlyophilized.

Bottles of co-lyophilized saccharide/protein material were brought toroom temperature and resuspended in 0.1M sodium phosphate buffer, pH7.0, to a final saccharide concentration of 20 mg/mL. The pH wasadjusted to 6.5 and then a 0.5 molar equivalent of sodiumcyanoborohydride was added. The reaction was incubated at 37° C. for 48hours. Following the cyanoborohydride incubation, the reaction mixturewas diluted with cold 5 mM succinate/0.9% saline buffer. Unreactedaldehydes were quenched by the addition of sodium borohydride andincubation at 23° C. for 3-6 hours. The reaction mixture was transferredthrough a 0.45-5 μm prefilter into a retentate vessel.

The reaction mixture was diafiltered 30× with 0.1M phosphate buffer (pH9), 20× with 0.15M phosphate butter (pH 6), and 20× with 5 mMsuccinate/0.9% saline. The retentate was filtered through a 0.2-μmfilter.

The conjugate solution was diluted to a saccharide target of 0.5 mg/mL,and then sterile filtered into FBC containers in a Class 100 hood. Theconjugate was stored at 2-8° C.

Characterization

Size exclusion chromatography media (CL-4B) was used to profile therelative molecular size distribution of the conjugate.

The identity of the conjugate was confirmed by the slot-blot assay usingspecific antisera.

The saccharide and protein concentrations were determined by theAnthrone and Lowry assays, respectively. The ratio of saccharide toprotein in the covalently bonded conjugate complex was obtained by thecalculation:

${Ratio} = \frac{{µg}\text{/}{mL}\mspace{14mu} {saccharide}}{{µg}\text{/}{mL}\mspace{14mu} {protein}}$

Example 5 Preparation of S. Pneumoniae Capsular Polysaccharide Serotype5

S. pneumoniae serotype 5 was obtained from Dr. Gerald Schiffman of theState University of New York, Brooklyn, N.Y. For preparation of the cellbank system, see Example 1. For fermentation, harvesting, purificationand characterization of the polysaccharide, see Example 1.

Alternate Fermentation Process

Cultures from the working cell bank were used to inoculate seed bottlescontaining a soy-based medium and a 10 mM sterile NaHCO₃ solution. Thebottles were incubated at 36° C.±2° C. without agitation until growthrequirements were met. A seed bottle was used to inoculate a seedfermentor containing soy-based medium and a 10 mM sterile NaHCO₃solution. A pH of about 7.0 was maintained with 3N NaOH. After thetarget optical density was reached, the seed fermentor was used toinoculate the production fermentor containing soy-based medium with a 10mM NaHCO₃ concentration. The pH was maintained with 3N NaOH. Thefermentation was terminated after cessation of growth or when theworking volume of the fermentor was reached. An appropriate amount ofsterile 12% sodium deoxycholate was added to the culture to obtain a0.12% concentration in the broth, to lyse the bacterial cells andrelease cell-associated polysaccharide. After lysing, the fermentorcontents were held, with agitation, for a time interval between 8 and 24hours at a temperature between 7° C. and 13° C. to assure that completecellular lysis and polysaccharide release had occurred. Agitation duringthis hold period prevented lysate sediment from settling on thefermentor walls and pH probe, thereby allowing the pH probe integrity tobe maintained. Next, the pH of the lysed culture broth was adjusted toapproximately pH 4.5 with 50% acetic acid. After a hold time withoutagitation, for a time interval between 12 and 24 hours at a temperaturebetween 15° C. and 25° C., a significant portion of the previouslysoluble proteins dropped out of solution as a solid precipitate withlittle loss or degradation of the polysaccharide, which remained insolution. The solution with the precipitate was then clarified bycontinuous flow centrifugation followed by depth filtration and 0.45 μmmicrofiltration.

Example 6 Preparation of Serotype 5 Pneumococcal Saccharide CRM₁₉₇Conjugate Activation and Conjugation

Containers of serotype 5 saccharide were thawed and combined in areaction vessel. To the vessel, 0.1M sodium acetate, pH 4.7, was addedfollowed by oxidation in the presence of sodium periodate by incubationfor 16-22 hours at 23° C.

The activation reaction mixture was concentrated and diafiltered 10×with WFI using a 100K MWCO membrane. The retentate was filtered througha 0.2 μm filter.

The serotype 5 activated saccharide was combined with CRM₁₉₇ at a ratioof 0.8:1. The combined saccharide/protein solution was filled into 100mL glass lyophilization bottles (50 mL target fill), shell-frozen at−75° C., and co-lyophilized.

Bottles of co-lyophilized material were brought to room temperature andresuspended in 0.1M sodium phosphate, pH 7.5, and sodiumcyanoborohydride was added. The reaction was incubated at 30° C. for 72hours, followed by a second addition of cyanoborohydride and incubatedat 30° C. for 20-28 hours.

Following the cyanoborohydride incubations, the reaction mixture wasdiluted 2-fold with saline and transferred through a 0.45-5 μm prefilterinto a retentate vessel. The reaction mixture was diafiltered 30× with0.01M phosphate buffer, pH 8, 20× with 0.15M phosphate buffer, pH 6, and20× with saline. The retentate was filtered through a 0.2 μm filter.

The conjugate solution was diluted to a saccharide target of 0.5 mg/mL,and then sterile filtered into FBC containers in a Class 100 hood. Theconjugate was stored at 2-8° C.

For the characterization of the conjugate, see Example 2.

Example 7 Preparation of S. Pneumoniae Capsular Polysaccharide Serotype6A

S. pneumoniae serotype 6A was obtained from Dr. Gerald Schiffman of theState University of New York, Brooklyn, N.Y. For preparation of the cellbank system, see Example 1. For fermentation, harvesting andpurification of the polysaccharide, see Example 1, except that duringpurification, the 30 kDa MWCO concentration step, prior to thechromatography step, is omitted.

Example 8 Preparation of Serotype 6A Pneumococcal Saccharide CRM₁₉₇Conjugate Activation and Conjugation

Serotype 6A polysaccharide is a high molecular weight polymer that hadto be reduced in size prior to oxidation. Containers of serotype 6Asaccharide were thawed and combined in a reaction vessel. To the vessel,2 M acetic acid was added to a final concentration of 0.1 M forhydrolysis for 1.5 hours at 60° C. The reaction was cooled to 23° C. andneutralization was performed by adjusting the reaction mixture with 1 MNaOH to pH 6. Oxidation in the presence of sodium periodate wasperformed by incubation at 23° C. for 14-22 hours.

The activation reaction mixture was concentrated and diafiltered 10×with WFI using a 100K MWCO membrane. The retentate was filtered througha 0.2 μm filter.

Serotype 6A was compounded with sucrose and filled into 100 mL glasslyophilization bottles (50 mL target fill) and shell-frozen at −75° C.and lyophilized.

Bottles of lyophilized material were brought to room temperature andresuspended in dimethylsulfoxide (DMSO) at a saccharide/protein ratio of1:1. After addition of sodium cyanoborohydride, the reaction mixture wasincubated at 23° C. for 18 hours. Following the cyanoborohydrideincubation, the reaction mixture was diluted with cold saline. Unreactedaldehydes were quenched by addition of sodium borohydride by incubationat 23° C. for 3-20 hours.

The diluted reaction mixture was transferred through a 5 μm prefilterinto a retentate vessel. The reaction mixture was diafiltered 10× with0.9% NaCl and 30× with succinate-buffered NaCl. The retentate wasfiltered through a 0.2 μm filter.

The conjugate solution was diluted to a saccharide target of 0.5 mg/mL,and then sterile filtered into FBC containers in a Class 100 hood. Theconjugate was stored at 2-8° C.

For the characterization of the conjugate, see Example 2.

Example 9 Preparation of S. Pneumoniae Capsular Polysaccharide Serotype7F

S. pneumoniae serotype 7F was obtained from Dr. Gerald Schiffman of theState University of New York, Brooklyn, N.Y. For preparation of the cellbank system, and for fermentation and harvesting of the polysaccharide,see Example 3. For an alternate fermentation and harvesting process, seethe alternate process described in Example 1.

Purification

The purification of the pneumococcal polysaccharide consisted of severalconcentration/diafiltration operations, precipitation/elution, columnchromatography, and depth filtration steps. All procedures wereperformed at room temperature unless otherwise specified.

Clarified broth from fermentor cultures of S. pneumoniae serotype 7Fwere concentrated and diafiltered using a 100 kDa MWCO filter.Diafiltration was accomplished using sodium phosphate buffer at neutralpH. Diafiltration removed the low molecular weight medium componentsfrom the higher molecular weight biopolymers such as nucleic acid,protein and polysaccharide.

Serotype 7F does not form a precipitate with HB. Instead, impuritieswere precipitated from the concentrated and diafiltered solution byadding the HB from a stock solution to a final concentration of 1% HB.The precipitate was captured on a depth filter and the filter wasdiscarded. The polysaccharide was contained in the filtrate.

Sodium iodide (NaI) was added to the polysaccharide solution from astock NaI solution to achieve a final concentration of 0.5% toprecipitate HB. The precipitate was removed by depth filtration. Thefiltrate contained the target polysaccharide. The precipitation vesseland the filter were rinsed with a NaCl/NaI solution and the rinses werecombined with the partially purified polysaccharide solution. The filterwas discarded. The polysaccharide was then filtered through a 0.2 μmfilter.

The polysaccharide solution was concentrated on a 30 kDa MWCOultrafilter and diafiltered with a sodium chloride solution.

The partially purified polysaccharide solution was further purified byfiltration through a depth filter impregnated with activated carbon.After filtration, the carbon filter was rinsed with a sodium chloridesolution. The rinse was combined with the polysaccharide solution, whichwas then filtered through a 0.2 μm filter.

The polysaccharide solution was concentrated on a 30 kDa MWCOultrafilter and adjusted with a 1M sodium phosphate buffer to achieve afinal concentration of 0.025M sodium phosphate. The pH was checked andadjusted to 7.0±0.2.

The ceramic hydroxyapatite (HA) column was equilibrated with sodiumphosphate buffer containing sodium chloride to obtain the appropriateconductivity (15 μS). The polysaccharide solution was then loaded ontothe column. Under these conditions, impurities bound to the resin andthe polysaccharide was recovered in the flow-through from the column.The polysaccharide was flushed through the column with buffer and wasfiltered through a 0.2 μm filter.

The polysaccharide solution was concentrated using a 30 kDa MWCO filter.The concentrate was then diafiltered with WFI.

The diafiltered polysaccharide solution was filtered through a 0.2 μmmembrane filter into stainless steel containers. Samples were removedfor release testing and the purified polysaccharide was stored at 2°-8°C.

For characterization of the polysaccharide, see Example 3.

Example 10 Preparation of Serotype 7F Pneumococcal Saccharide—CRM₁₉₇Conjugate Activation and Conjugation

Oxidation in the presence of sodium periodate was performed byincubation for 16-24 hrs at 23° C.

The activation reaction mixture was concentrated and diafiltered 10×with 10 mM NaOAc, pH 4.5, using a 100K MWCO membrane. The retentate wasfiltered through a 0.2 μm filter.

Serotype 7F was filled into 100 mL glass lyophilization bottles (50 mLtarget fill) and shell-frozen at −75° C. and lyophilized.

Bottles of lyophilized serotype 7F and CRM₁₉₇ were brought to roomtemperature and resuspended in DMSO at a saccharide/protein ratio of1.5:1. After the addition of sodium cyanoborohydride, the reaction wasincubated at 23° C. for 8-10 hours. Unreacted aldehydes were quenched bythe addition of sodium borohydride by incubation at 23° C. for 16 hours.

The reaction mixture was diluted 10-fold with cold saline andtransferred through a 5 μm prefilter into a retentate vessel. Thereaction mixture was diafiltered 10× with 0.9% saline and 30× withsuccinate-buffered saline. The retentate was filtered through a 0.2 μmfilter.

The conjugate solution was diluted to a saccharide target of 0.5 mg/mL0.9% saline, and then sterile filtered into FBC containers in a Class100 hood. The conjugate was stored at 2-8° C.

For characterization of the conjugate, see Example 4.

Example 11 Preparation of S. Pneumoniae Capsular Polysaccharide Serotype19A

S. pneumoniae serotype 19A was obtained from Dr. Gerald Schiffman of theState University of New York, Brooklyn, N.Y. For preparation of the cellbank system, see Example 1. For fermentation, harvesting andpurification of the polysaccharide, see Example 7. For characterization,see Example 3.

Example 12 Preparation of Serotype 19A Pneumococcal Saccharide CRM₁₉₇Conjugate Activation and Conjugation

Containers of serotype 19A saccharide were thawed and combined in areaction vessel. Sodium acetate was added to 10 mM (pH 5.0) andoxidation was carried out in the presence of sodium periodate byincubation for 16-24 hrs at 23° C.

The activation reaction mixture was concentrated and diafiltered 10×with 10 mM acetate, pH 5.0, using a 100K MWCO membrane. The retentatewas filtered through a 0.2 μm filter.

The activated saccharide was compounded with sucrose followed by theaddition of CRM₁₉₇. The serotype 19A activated saccharide and CRM₁₉₇mixture (0.8:1 ratio) was filled into 100 mL glass lyophilizationbottles (50 mL target fill) and shell-frozen at −75° C. and lyophilized.

Bottles of lyophilized material were brought to room temperature andresuspended in DMSO. To the saccharide/protein mixture, sodiumcyanoborohydride (100 mg/ml) was added. The reaction was incubated at23° C. for 15 hours. Following the cyanoborohydride incubation,unreacted aldehydes were quenched by the addition of sodium borohydrideby incubation at 23° C. for 3-20 hours.

The reaction mixture was diluted 10-fold with cold saline andtransferred through a 5 μm prefilter into a retentate vessel. Thereaction mixture was diafiltered 10× with 0.9% NaCl, 0.45-μm filtered,and 30× with diafiltration using 5 mM succinate/0.9% NaCl buffer, pH 6.The retentate was filtered through a 0.2 μm filter.

The conjugate solution was diluted to a target of 0.5 mg/mL using 5 mMsuccinate/0.9% saline, and then sterile filtered into FBC containers ina Class 100 hood. The conjugate was stored at 2-8° C.

For characterization of the conjugate, see Example 4.

Example 13 Preparation of S. Pneumoniae Capsular PolysaccharideSerotypes 4, 6B, 9V, 14, 18C, 19F and 23F Preparation of the S.pneumoniae Seed Culture

S. pneumoniae serotypes 4, 6B, 9V, 18C, 19F and 23F were obtained fromDr. Gerald Schiffman, State University of New York, Brooklyn, N.Y. S.pneumoniae serotype 14 was obtained from the ATCC, strain 6314.

Separately, one vial of each of the desired serotypes of Streptococcuspneumoniae was used to start a fermentation batch. Two bottlescontaining a soy-based medium and phenol red were adjusted to a pH rangeof 7.4±0.2 using sodium carbonate, and the required volume of 50%dextrose/1% magnesium sulfate solution was then added to the bottles.The two bottles were inoculated with different amounts of seed. Thebottles were incubated at 36°±2° C. until the medium turned yellow.Following incubation, samples were removed from each bottle and testedfor optical density (OD) (0.3 to 0.9) and pH (4.6 to 5.5). One of thetwo bottles was selected for inoculation of the seed fermentor.

Soy-based medium was transferred to the seed fermentor and sterilized.Then a volume of 50% dextrose/1% magnesium sulfate solution was added tothe fermentor. The pH and agitation of the seed fermentor were monitoredand controlled (pH 6.7 to 7.4). The temperature was maintained at 36°±2°C. The seed inoculum (bottle) was aseptically connected to the seedfermentor and the inoculum was transferred. The fermentor was maintainedin pH control and samples were periodically removed and tested for ODand pH. When the desired OD of 0.5 at 600 nm was reached, theintermediate fermentor was inoculated with the fermentation broth fromthe seed fermentor.

Soy-based medium was transferred to the intermediate fermentor andsterilized. Then a volume of 50% dextrose/1% magnesium sulfate solutionwas added to the fermentor. The pH and agitation of the intermediatefermentor were monitored and controlled (pH 6.7 to 7.4). The temperaturewas maintained at 360±2° C. The contents of the seed fermentor weretransferred to the intermediate fermentor. The fermentor was maintainedin pH control and samples were periodically removed and tested for ODand pH. When the desired OD of 0.5 at 600 nm was reached, the productionfermentor was inoculated with the fermentation broth from theintermediate fermentor.

Soy-based medium was transferred to the production fermentor andsterilized. Then a volume of 50% dextrose/1% magnesium sulfate solutionwas added to the fermentor. The pH and agitation of the productionfermentor were monitored and controlled (pH 6.7 to 7.4). The temperaturewas maintained at 36°+2° C. The fermentor was maintained in pH controland samples were periodically removed and tested for OD and pH, untilthe fermentation was complete.

Deoxycholate sodium was added to the fermentor to a final concentrationof approximately 0.12% w/v. The culture was mixed for a minimum ofthirty minutes and the temperature set point was reduced to 10° C. Theculture was incubated overnight and following confirmation ofinactivation, the pH of the culture was adjusted to between 6.4 and 6.8,as necessary, with 50% acetic acid. The temperature of the fermentor wasincreased to 20°±5° C. and the contents were transferred to theclarification hold tank.

The contents of the clarification hold tank (including the cellulardebris) were processed through a centrifuge at a flow rate between 25and 600 liters per hour (except Serotype 4, wherein the cell debris wasdiscarded and the flow rate tightened to between 25 and 250 liters perhour). Samples of the supernatant were removed and tested for OD. Thedesired OD during the centrifugation was ≦0.15.

Initially, the supernatant was recirculated through a depth filterassembly until an OD of 0.05±0.03 was achieved. Then the supernatant waspassed through the depth filter assembly and through a 0.45 μm membranefilter to the filtrate hold tank.

Subsequently, the product was transferred through closed pipes to thepurification area for processing.

All of the above operations (centrifugation, filtration and transfer)were performed between 10° C. to 30° C.

For an alternate fermentation and harvesting process for serotypes 4 and6B, see the alternate process described in Example 1.

Purification

The purification of each pneumococcal polysaccharide consisted ofseveral concentration/diafiltration operations, precipitation/elution,column chromatography, and depth filtration steps. All procedures wereperformed at room temperature unless otherwise specified.

Clarified broth from the fermentor cultures of the desired S. pneumoniaeserotype was concentrated and diafiltered using a 100 kDa MWCO filter.Diafiltration was accomplished using sodium phosphate buffer at pH<9.0.Diafiltration removed the low molecular weight medium components fromthe higher molecular weight biopolymers such as nucleic acid, proteinand polysaccharide.

The polysaccharide was precipitated from the concentrated anddiafiltered solution by adding HB from a stock solution to give a finalconcentration of 1% HB (w/v) (except Serotype 23F, which had a finalconcentration of 2.5%). The polysaccharide/HB precipitate was capturedon a depth filter and the filtrate was discarded. (Note: Serotype 14does not precipitate; therefore the filtrate was retained.) Thepolysaccharide precipitate was resolubilized and eluted by recirculatinga sodium chloride solution through the precipitate-containing depthfilter. The filters were then rinsed with additional sodium chloridesolution.

Sodium iodide (NaI) was added to the polysaccharide solution from astock NaI solution to achieve a final concentration of 0.5% toprecipitate HB (except for Serotype 6B, which had a final concentrationof 0.25%). The precipitate was removed by depth filtration. The filtratecontained the target polysaccharide. The filter was discarded. Thepolysaccharide was then filtered through a 0.2 μm filter.

The polysaccharide solution was concentrated on a 30 kDa MWCOultrafilter and diafiltered with a sodium chloride solution.

The partially purified polysaccharide solution was further purified byfiltration through a depth filter impregnated with activated carbon.After filtration, the carbon filter was rinsed with a sodium chloridesolution. The rinse was combined with the polysaccharide solution, whichwas then filtered through a 0.2 μm filter.

The polysaccharide solution was concentrated on a 30 kDa MWCOultrafilter and the filter was rinsed with a sodium chloride solution.The pH was checked and adjusted to 7.0±0.3.

The ceramic hydroxyapatite (HA) column was equilibrated with sodiumphosphate buffer containing sodium chloride until the pH is 7.0±0.3 andthe conductivity was 26±4 μS. The polysaccharide solution was thenloaded onto the column. Under these conditions, impurities bound to theresin and the polysaccharide was recovered in the flow through from thecolumn. The polysaccharide solution was filtered through a 0.2 μmfilter.

The polysaccharide solution was concentrated using a 30 kDa MWCO filter.The concentrate was then diafiltered with WFI until the conductivity was<15 μS.

The diafiltered polysaccharide solution was filtered through a 0.2 μmmembrane filter into bulk containers and stored at 2-8° C.

Example 14 Preparation of Pneumococcal Saccharide CRM₁₉₇ Conjugates forSerotypes 4, 6B, 9V, 14, 18C, 19F and 23F Activation Process

The different serotype saccharides follow different pathways foractivation (hydrolysis or no hydrolysis prior to activation) andconjugation (aqueous or DMSO reactions) as described in this example.

Polysaccharide was transferred from the bulk containers to the reactorvessel. The polysaccharide was then diluted in WFI and sodium phosphateto a final concentration range of 1.6-2.4 mg/mL.

Step 1.

For serotypes 6B, 9V, 14, 19F and 23F, pH was adjusted to pH 6.0±0.3.For serotype 4, hydrochloric acid (0.01 M final acid concentration) wasadded and the solution was incubated for 25-35 minutes at 450±2° C.Hydrolysis was stopped by cooling to 21-25° C. and adding 1M sodiumphosphate to a target of pH 6.7±0.2. An in-process test was done toconfirm an appropriate level of depyruvylation.

For serotype 18C, glacial acetic acid (0.2 M final acid concentration)was added and the solution was incubated for 205-215 minutes at 94°±2°C. Temperature was then decreased to 21-25° C. and 1-2 M sodiumphosphate was added to a target of pH 6.8±0.2.

Step 2: Periodate Reaction

The required sodium periodate molar equivalents for pneumococcalsaccharide activation was determined using total saccharide content(except for serotype 4). For serotype 4, a ratio of 0.8-1.2 moles ofsodium periodate per mole of saccharide was used. With thorough mixing,the oxidation reaction was allowed to proceed between 16 to 20 hours at21-25° C. for all serotypes except 19F for which the temperature was≦15° C.

Step 3: Ultrafiltration

The oxidized saccharide was concentrated and diafiltered with WFI (0.01M sodium phosphate buffer pH 6.0 for serotype 19F) on a 100 kDa MWCOultrafilter (5 kDa ultrafilter for 18C). The permeate was discarded andthe retentate was filtered through a 0.22 μm filter.

Step 4: Lyophilization

For serotypes 4, 9V, and 14 the concentrated saccharide was mixed withCRM₁₉₇ carrier protein, filled into glass bottles, shell-frozen andstored at ≦−65° C. The frozen concentrated saccharide-CRM₁₉₇ waslyophilized and then stored at −25°±5° C.

For serotypes 6B, 19F, and 23F a specified amount of sucrose was addedwhich was calculated to achieve a 5%±3% sucrose concentration in theconjugation reaction mixture. Serotype 18C did not require sucroseaddition. The concentrated saccharide was then filled into glassbottles, shell-frozen and stored at ≦−65° C. The frozen concentratedsaccharide was lyophilized and then stored at −25°±5° C.

Conjugation Process

Two conjugation processes were used: aqueous conjugation for serotypes4, 9V, 14 and 18C, and DMSO conjugation for serotypes 6B, 19F and 23F.

Aqueous Conjugation Step 1: Dissolution

For serotypes 4, 9V and 14, the lyophilized activated saccharide-CRM₁₉₇mixture was thawed and equilibrated at room temperature. The lyophilizedactivated saccharide-CRM₁₉₇ was then reconstituted in 0.1M sodiumphosphate buffer at a typical ratio of:

1 L of buffer per 16-24 g of saccharide for serotype 4 and 9V

1 L of buffer per 6-10 g of saccharide for serotype 14

The reaction mixture was incubated at 370±2° C. until total dissolutionfor the serotype 9V and at 23°±2° C. for serotypes 4 and 14.

For serotype 18C, the lyophilized saccharide was reconstituted in asolution of CRM₁₉₇ in 1M dibasic sodium phosphate at a typical ratio of0.11 L of sodium phosphate per 1 L of CRM₁₉₇ solution. The reactionmixture (8-12 g/L saccharide concentration) was incubated at 23°±2° C.until total dissolution.

The pH was tested as an in-process control at this stage.

Step 2: Conjugation Reaction

For serotypes 4 and 9V, the conjugation reaction was initiated by addingthe sodium cyanoborohydride solution (100 mg/mL) to achieve 1.0-1.4moles sodium cyanoborohydride per mole of saccharide. The reactionmixture was incubated for 44-52 hours at 37°±2° C. The temperature wasthen reduced to 23°±2° C. and sodium chloride 0.9% was added to thereactor. Sodium borohydride solution (100 mg/mL) was added to achieve1.8-2.2 molar equivalents of sodium borohydride per mole saccharide. Themixture was incubated for 3-6 hours at 23°±2° C. The mixture was dilutedwith sodium chloride 0.9% and the reactor was rinsed. The dilutedconjugation mixture was filtered using a 1.2 μm pre-filter into aholding vessel.

For serotypes 14 and 18C, the conjugation reaction was initiated byadding the cyanoborohydride solution (100 mg/mL) to achieve 1.0-1.4moles of sodium cyanoborohydride per mole of saccharide. The reactionmixture was incubated for 12-24 hours at 23°±2° C. The temperature wasincreased to 37°±2° C. and the reaction was incubated for 72-96 hours.The temperature was then reduced to 23°±2° C. and 0.9% sodium chloridewas added to the reactor. Sodium borohydride solution (100 mg/mL) wasadded to achieve 1.8-2.2 molar equivalents of sodium borohydride permole of saccharide. The mixture was incubated for 3-6 hours at 23°±2° C.The mixture was diluted with 0.9% sodium chloride and the reactor wasrinsed. The diluted conjugation mixture was then filtered using a 1.2 μmpre-filter into a holding vessel.

Step 3: Ultrafiltration 100 kDa

The diluted conjugation mixture was concentrated and diafiltrated on a100 kDa MWCO ultrafilter with either a minimum of 15 volumes (serotype4) or 40 volumes (serotypes 9V, 14, and 18C) of 0.9% sodium chloride.

The permeate was discarded.

For serotype 4, the retentate was filtered through a 0.45 μm filter.

An in-process control (saccharide content) was performed at this step.

Step 4: HA Column Purification

This step was only performed for the serotype 4 conjugate.

The HA column was first neutralized using 0.5M sodium phosphate buffer(pH 7.0±0.3) and then equilibrated with 0.9% sodium chloride. Thefiltered retentate (serotype 4) was loaded onto the column at a flowrate of 1.0 L/min. The column was washed with 0.9% sodium chloride at aflow rate of ≦2.0 L/min. The product was then eluted with 0.5M sodiumphosphate buffer at a flow rate of ≦2.0 L/min.

The HA fraction was then concentrated and diafiltered on a 100 kDa MWCOmembrane with a minimum of 20 volumes of 0.9% sodium chloride. Thepermeate was discarded.

Step 5: Sterile Filtration

The retentate after the 100 kDa MWCO diafiltration was filtered througha 0.22 μm filter. In-process controls (saccharide content, free protein,free saccharide and cyanide) were performed on the filtered product.In-process controls on filtered retentate were performed to determinewhether additional concentration, diafiltration, and/or dilution wereneeded to meet FBC targets. These and additional tests were repeated inFBC samples.

As necessary, the filtered conjugate was diluted with 0.9% sodiumchloride in order to achieve a final concentration of less than 0.55g/L. Release tests for saccharide content, protein content andsaccharide:protein ratio were performed at this stage.

Finally, the conjugate was filtered (0.22 μm) and filled into 10 Lstainless steel canisters at a typical quantity of 2.64 g/canister. Atthis stage, yield, saccharide content, protein content, pH,saccharide:protein ratio and lysine content were performed as in-processcontrols. Release testing (appearance, free protein, free saccharide,endotoxin, molecular size determination, residual cyanide, saccharideidentity, CRM₁₉₇ identity) was performed at this stage.

DMSO Conjugation Step 1: Dissolution

The lyophilized activated saccharide serotypes 6B, 19F, 23F and thelyophilized CRM₁₉₇ carrier protein were equilibrated at room temperatureand reconstituted in DMSO. The dissolution concentration typicallyranged from 2-3 grams of saccharide (2-2.5 g protein) per liter of DMSO.

Step II: Conjugation Reaction

The activated saccharide and CRM₁₉₇ carrier protein were mixed for 60-75minutes at 23°±2° C. at a ratio range of 0.6 g-1.0 g saccharide/g CRM₁₉₇for serotypes 6B and 19F or 1.2 to 1.8 g saccharide/g CRM₁₉₇ forserotype 23F.

The conjugation reaction was initiated by adding the sodiumcyanoborohydride solution (100 mg/mL) at a ratio of 0.8-1.2 molarequivalents of sodium cyanoborohydride to one mole activated saccharide.WFI was added to the reaction mixture to a target of 1% (v/v) and themixture was incubated for over 40 hours at 23°±2° C.

Sodium borohydride solution, 100 mg/mL (typical 1.8-2.2 molarequivalents sodium borohydride per mole activated saccharide) and WFI(target 5% v/v) were added to the reaction and the mixture was incubatedfor 3-6 hours at 23°±2° C. This procedure reduced any unreactedaldehydes present on the saccharides. Then the reaction mixture wastransferred to a dilution tank containing 0.9% sodium chloride at <15°C.

Step III: 100 kDa Ultrafiltration

The diluted conjugate mixture was filtered through a 1.2 μm filter andconcentrated and diafiltered on a 100 kDa MWCO membrane with a minimumof 15 volumes of 0.9% sodium chloride (0.01M sodium phosphate/0.05M NaClbuffer was used for serotype 23F). The permeate was discarded. Theretentate was filtered through a 0.45 μm filter. An in-processsaccharide content sample was taken at this stage.

Step IV: DEAE Column Purification

This step was only performed for serotype 23F.

The DEAE column was equilibrated with 0.01M sodium phosphate/0.05Msodium chloride buffer. The filtered retentate (serotype 23F) was loadedonto the column and washed with 0.01M sodium phosphate/0.05M sodiumchloride buffer. The column was then washed with 0.01M sodiumphosphate/0.9% NaCl buffer. The product was then eluted with 0.01Msodium phosphate/0.5M sodium chloride buffer.

Step V: 100 kDa Ultrafiltration

The retentate from 6B and 19F was concentrated and diafiltered with atleast 30 volumes of 0.9% sodium chloride. The permeate was discarded.

The eluate from serotype 23F was concentrated and diafiltered with aminimum of 20 volumes of 0.9% sodium chloride. The permeate wasdiscarded.

Step VI: Sterile Filtration

The retentate after the 100 kDa MWCO diafiltration was filtered through0.22 μm filter. In-process controls (saccharide content, free protein,free saccharide, residual DMSO and residual cyanide) were performed onthe filtered product. In-process controls on filtered retentate wereperformed to determine whether additional concentration, diafiltration,and/or dilution were needed to meet FBC targets. These and additionaltests were repeated in FBC samples.

As necessary, the filtered conjugate was diluted with 0.9% sodiumchloride to achieve a final concentration of less than 0.55 g/L. Releasetests for saccharide content, protein content and saccharide:proteinratio were performed at this stage.

Finally, the conjugate was filtered (0.22 μm) and filled into 10 Lstainless steel canisters at a quantity of 2.64 g/canister. At thisstage, yield, saccharide content, protein content, pH,saccharide:protein ratio and lysine content were performed as in-processcontrols. Release testing (appearance, free protein, free saccharide,endotoxin, molecular size determination, residual cyanide, residualDMSO, saccharide identity and CRM₁₉₇ identity) was performed at thisstage.

Example 15 Formulation of a Multivalent Pneumococcal Conjugate Vaccine

The final bulk concentrates of the 13 conjugates contain 0.85% sodiumchloride. Type 3, 6A, 7F and 19A bulk concentrates also contain 5 mMsodium succinate buffer at pH 5.8. The required volumes of bulkconcentrates were calculated based on the batch volume and the bulksaccharide concentrations. After 80% of the 0.85% sodium chloride(physiological saline) and the required amount of succinate buffer wereadded to the pre-labeled formulation vessel, bulk concentrates wereadded. The preparation was then sterile filtered through a 0.22 μmmembrane into a second container by using a Millipore Durapore membranefilter unit. The first container was washed with the remaining 20% of0.85% sodium chloride and the solution was passed through the samefilter and collected into the second container. The formulated bulk wasmixed gently during and following the addition of bulk aluminumphosphate. The pH was checked and adjusted if necessary. The formulatedbulk product was stored at 2-8° C.

The formulated bulk product was filled into Type 1 borosilicate glasssyringes obtained from Becton Dickinson. The vaccine was monitored atregular intervals for turbidity to ensure the uniformity of the fillingoperation. The filled vaccine (Final Product) was stored at 2-8° C.

Example 16 Immunogenicity of the 13-Valent Conjugate Vaccine

To date, the preclinical studies performed on the 13vPnC vaccine havebeen in rabbits. Studies #HT01-0021 and #HT01-0036 were designed toindependently examine the effect of chemical conjugation of capsularpolysaccharides (PSs) from S. pneumoniae to CRM₁₉₇ and the effect ofaluminum phosphate (AIPO₄) adjuvant on the immune response to the 13vPnCvaccine in rabbits. These effects were characterized by antigen-specificELISA for serum IgG concentrations and for antibody function byopsonophagocytic assay (OPA).

Study #HT01-0021

Study #HT01-0021 examined the ability of the 13vPnC vaccine with AIPO₄adjuvant to elicit vaccine serotype-specific immune responses. Thepneumococcal serotypes represented in the 13vPnC vaccine include types1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Secondaryobjectives included an evaluation of the kinetics and duration of theantibody response. New Zealand White rabbits were immunizedintramuscularly at week 0 and week 2 with the planned human clinicaldose of each polysaccharide (2 μg of each PS, except 4 μg of 6B)formulated with or without AIPO₄ (100 μg/dose). Sera were collected atvarious time points. Serotype specific IgG was measured by ELISA andfunctional activity was assessed by OPA.

Table 3 shows the geometric mean titer (GMT) achieved in pooled serumsamples, following two doses of the 13vPnC vaccine. A ratio of the IgGGMTs was used to compare responses from week 4 to week 0. These datademonstrate that the inclusion of AIPO₄ in the 13vPnC formulationelicited higher levels of IgG antibody in comparison to the same vaccinewithout adjuvant. Although the antibody responses were greater whenAIPO₄ was included in the formulation, these increases were notstatistically significant.

Functional antibody responses were also assessed in rabbits followingimmunization with the two 13vPnC formulations (Table 4). When comparingvaccine formulations with or without adjuvant, higher OPA GMTs wereobserved in the 13vPnC+AIPO₄ vaccine treatment group. OPA titers weredetected in week 4 serum pools to all vaccine serotypes in both groups.For the majority of the serotypes, OPA titers measured at week 4 were atleast 4-fold higher than those at week 0 (baseline).

The kinetic responses to each of the 13vPnC vaccine serotypes wereevaluated from serum pools of both treatment groups. IgG titers to eachserotype were measured from blood draws at week 0 and weeks 1, 2, 3, 4,8, 12, 26, and 39 and then compared. With the exception of serotype 1,antibody responses in animals receiving adjuvanted vaccine were superiorto those that received non-adjuvanted vaccine and peaked at week 2 ofthe immunization schedule (data not shown).

Overall, the data indicate that the 13vPnC vaccine formulated withaluminum phosphate is immunogenic in rabbits, eliciting substantialantibody responses to the pneumococcal capsular polysaccharidescontained in the vaccine and these responses are associated withfunctional activity. The responses observed to the seven core serotypesfollowing immunization with 13vPnC+AIPO₄ are consistent with historicalresponses of rabbits to the heptavalent formulation.

TABLE 3 Rabbit IgG Immune Responses (GMTs) Following Immunization withTwo Doses of 13-valent Pneumococcal Glycoconjugate Diluent with ALPO₄^(a) 13vPnC^(a) 13vPnC + ALPO₄ ^(a) Ratio Week 4 Ratio Week 4 RatioSerotype Week 0 Week 4 Wk4:Wk0 Week 0 (95% CI) Wk4:Wk0 Week 0 (95% CI)Wk4:Wk0  1 <100 <100 1.0 50 5,926 119 50 11,091 222 (2,758-12,733)(5,327-23,093)  3 <100 <100 1.0 50 6,647 133 58 16,443 284(2,773-15,932) (7,096-38,106)  4 <100 <100 1.0 50 13,554  271 50 29,183584 (8,031-22,875) (15,342-55,508)   5 134 <100 0.4 50 5,859 117 5016,714 334 (2,450-14,009) (6,959-40,140)  6A 141 <100 0.4 74 22,415  30383 63,734 768 (11,987-41,914)  (21,141-192,146)  6B <100 <100 1.0 578,108 142 54 23,505 435 (3,564-18,444) (11,286-48,955)   7F 3,859 5790.2 171 43,591  444 143 84,888 496 (26,931-70,557)  (46,445-155,151)  9V289 995 3.4 205 15,780  125 208  43,331^(b) 217 (7,193-34,616)(23,256-71,510)  14 437 177 0.4 61 6,906 113 70 16,076 322(3,416-13,962) (9,649-26,785) 18C <100 <100 1.0 50 21,283  426 50 35,040701 (15,770-28,725)  (24,708-49,692)  19A <100 <100 1.0 121 113,599  939144 280,976  1,951 (54,518-236,707) (119,587-660,167)  19F <100 <100 1.050 14,365  287 50 24,912 498 (7,346-28,090) (9,243-67,141) 23F <100 <1001.0 50 5,323 106 50 15,041 301 (1,894-14,962) (4,711-48,018) ^(a)GMTs ofpooled sera consisted of equal volumes of serum from each individualrabbit within a group ^(b)Statistically different (p = 0.022) fromtreatment group without ALPO₄

TABLE 4 S. pneumoniae OPA GMTs for NZW Rabbit Serum Pools FollowingImmunization with Two Doses of 13-valent Pneumococcal Glycoconjugate13vPnC^(a) 13vPnC + ALPO₄ ^(a) Ratio Ratio Serotype Week 0 Week 4Wk4:Wk0 Week 0 Week 4 Wk4:Wk0  1 <8 64 16 <8 64 16  3 <8 8 2 <8 16 4  4<8 16 4 <8 32 8  5 <8 128 32 <8 512 128  6A 8 128 16 8 512 64  6B <8 25664 8 1,024 128  7F 8 64 8 8 128 16  9V 8 64 8 8 128 16 14 16 32 2 16 322 18C 8 256 32 <8 256 64 19A <8 256 64 <8 1,024 256 19F <8 128 32 <8 512128 23F 8 64 8 <8 256 64 ^(a)Pools consisted of equal volumes of serumfrom individual rabbits within a treatment group (n = 12)

Study #HT01-0036

Study #HT01-0036 compared rabbit immune responses to the polysaccharides(PSs) contained in the vaccine, after immunization with the 13vPnCvaccine with or without conjugation to the CRM₁₉₇ protein. New ZealandWhite rabbits were immunized intramuscularly at week 0 and week 2 with adose of 2.2 μg of each PS (except 4.4 μg of 6B). Animals received one ofthree vaccine preparations: (a) 13vPnC (PS directly conjugated toCRM₁₉₇), (b) 13vPnPS, (free PS) or (c) 13vPnPS+CRM₁₉₇ (free PS mixedwith CRM₁₉₇). All vaccine preparations contained AIPO₄ as the adjuvantat 125 μg/dose.

Serotype specific immune responses for all vaccine preparations wereevaluated in an IgG ELISA and complement-mediated OPA measuringfunctional antibody. The immune responses were compared between thetreatment groups.

Table 5 presents GMT data obtained from week 4 bleeds analyzed inantigen specific IgG ELISAs. Additional analyses show the ratio of GMTvalues at week 4 to week 0. The data indicate that the conjugate vaccinepreparation elicited greater serum IgG titers than free PS or freePS+CRM₁₉₇ vaccine. With the exception of S. pneumoniae type 14, the13vPnC vaccine was able to induce functional antibodies to therepresentative strains of S. pneumoniae in an OPA (Table 6). After twoimmunizations with either the 13vPnPS or 13vPnPS+CRM₁₉₇ vaccine, neithercould induce OPA titers ≧8-fold at week 4 relative to week 0 for 10 outof the 13 serotypes measured (Table 6).

In conclusion, these results indicate that conjugation of the 13-valentpneumococcal vaccine polysaccharides produces higher serum IgG titersand overall greater functional antibody activity than seen with freepolysaccharide alone or mixed with unconjugated CRM₁₉₇.

TABLE 5 Rabbit IgG Responses (GMTs) to PnPS by ELISA FollowingImmunization with Two Doses of 13-valent Pneumococcal Glycoconjugate13vPnPS = CRM₁₉₇ 13vPnPS (free PS) (PS mixed with CRM₁₉₇) 13VPnC Week 4Ratio Week 4 Ratio Week 4 Ratio Serotype Week 0 (95% CI) Wk4:Wk0 Week 0(95% CI) Wk4:Wk0 Week 0 (95% CI) Wk4:Wk0  1 378 2,290   5.8 395 1,959  5.0 472 35,970 76.2   (843-5,790) (809-4,739) (29,130-44,417)  3 57 2404.2 89 163 1.8 50 10,414 208.3  (64-908) (74-358)  (10,414-16,676)  4 50379 7.6 50 607 12.1 50 12,890 257.8 (150-959) (313-1,178) (9,117-18,224)  5 343 226 4.5 50 321 6.4 50 35,264 705.3 (113-450)(147-701)   (24,467-50,824)  6A 154 466 3.0 98 210 2.1 163 234,245 1,437.1 (316-688) (95-464)  (167,152-328,283)  6B 63 727 11.6 62 74512.0 131 33,599 256.5   (384-1,375) (384-1,440) (22,934-49,222)  7F 50 61 1.2 50  72 1.4 50 35,702 714.0 (39-95) (47-111)  (24,350-52,347)  9V50 104 2.1 55 169 3.0 50 50,033 1,000.7  (48-195) (74-390) (34,765-72,007) 14 66 298 4.5 50 195 3.9 50 20,121 402.4 (117-757)(71-535)  (12,087-32,138) 18C 89 1,555   17.5 66 761 11.5 101 71,451707.4   (655-3,688) (300-1,935)  (32,745-124,641) 19A 50  89 1.8 50  801.6 50 23,485 469.7  (44-179) (39-163)  (12,857-42,723) 19F 61 1,362  22.3 61 991 16.3 67 19,358 288.9   (559-3,317) (370-2,654)(12,553-33,173) 23F 73 1,085   14.9 121 638 5.3 68 45,972 676.1  (487-2,420) (311-1,311) (25,134-84,089)

TABLE 6 S. pneumoniae OPA Titers for Rabbit Serum Pools FollowingImmunization with Two Doses of 13-valent Pneumococcal Vaccines OPATiters 13vPnPS + CRM₁₉₇ (free PS mixed with No 13vPnPS (free PS) CRM₁₉₇)13vPnC Treatment Ratio Ratio Ratio Serotype Week 0^(a) Week 4 Wk4:Wk0Week 4 Wk4:Wk0 Week 4 Wk4:Wk0  1 4 16 4 16 4 8 32  3 4 4 1 4 1 4 8  4 44 1 4 1 4 64  5 4 32 8 16 4 16 64  6A 8 64 8 32 4 32 664  6B 8 64 8 32 432 32  7F 16 32 2 16 1 16 16  9V 16 16 1 32 2 32 8 14 16 16 1 16 1 16 218C 4 16 4 16 4 8 64 19A 8 8 1 8 1 16 64 19F 4 4 1 4 1 8 64 23F 16 32 216 1 32 32 ^(a)Used as week 0 values for all groups

It should be understood that the foregoing discussion and examplesmerely present a detailed description of certain embodiments. Ittherefore should be apparent to those of ordinary skill in the art thatvarious modifications and equivalents can be made without departing fromthe spirit and scope of the invention.

All journal articles, other references, patents and patent applicationsthat are identified in this patent application are incorporated byreference in their entirety.

REFERENCES

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1. A method for making an immunogenic conjugate comprising Streptococcuspneumoniae serotype 1 polysaccharide covalently linked to a carrierprotein, the method comprising: (a) reacting purified serotype 1polysaccharide with an alkaline pH buffer resulting in a partiallyde-O-acetylated serotype 1 polysaccharide; (b) reacting the partiallyde-O-acetylated serotype 1 polysaccharide with a mild acid resulting ina neutralized, partially de-O-acetylated serotype 1 polysaccharide; (c)reacting the neutralized, partially de-O-acetylated serotype 1polysaccharide with an oxidizing agent resulting in an activatedserotype 1 polysaccharide; (d) compounding the activated serotype 1polysaccharide with a carrier protein; (e) reacting the compounded,activated serotype 1 polysaccharide and carrier protein with a reducingagent resulting in a serotype 1 polysaccharide:carrier proteinconjugate; and (f) capping unreacted aldehydes in the serotype 1polysaccharide:carrier protein conjugate resulting in an immunogenicconjugate comprising Streptococcus pneumoniae serotype 1 polysaccharidecovalently linked to a carrier protein.
 2. The method of claim 1,further comprising purifying the activated serotype 1 polysaccharideprior to compounding with the carrier protein.
 3. The method of claim 1,further comprising co-lyophilizing the compounded, activated serotype 1polysaccharide and carrier protein prior to reacting with the reducingagent.
 4. The method of claim 1, further comprising purifying theimmunogenic conjugate.
 5. The method of claim 1, wherein the alkaline pHbuffer is a bicarbonate/carbonate buffer.
 6. The method of claim 1,wherein the mild acid is acetic acid.
 7. The method of claim 1, whereinthe mild acid is a low ionic strength mineral acid.
 8. The method ofclaim 7, wherein the low ionic strength mineral acid is hydrochloricacid.
 9. The method of claim 1, wherein the oxidizing agent is sodiumperiodate.
 10. The method of claim 1, wherein the carrier protein isCRM₁₉₇.
 11. The method of claim 1, wherein the reducing agent is sodiumcyanoborohydride.
 12. The method of claim 1, wherein capping unreactedaldehydes comprises reacting the serotype 1 polysaccharide:carrierprotein conjugate with sodium borohydride.
 13. A method for making animmunogenic conjugate comprising Streptococcus pneumoniae serotype 1polysaccharide covalently linked to a carrier protein, the methodcomprising: (a) reacting purified serotype 1 polysaccharide with abicarbonate/carbonate buffer resulting in a partially de-O-acetylatedserotype 1 polysaccharide; (b) reacting the partially de-O-acetylatedserotype 1 polysaccharide with acetic acid resulting in a neutralized,partially de-O-acetylated serotype 1 polysaccharide; (c) reacting theneutralized, partially de-O-acetylated serotype 1 polysaccharide withsodium periodate resulting in an activated serotype 1 polysaccharide;(d) purifying the activated serotype 1 polysaccharide; (e) compoundingthe activated serotype 1 polysaccharide with a carrier protein; (f)co-lyophilizing the compounded, activated serotype 1 polysaccharide andcarrier protein; (g) reacting the co-lyophilized, activated serotype 1polysaccharide and carrier protein with sodium cyanoborohydrideresulting in a serotype 1 polysaccharide:carrier protein conjugate; and(h) capping unreacted aldehydes in the serotype 1 polysaccharide:carrierprotein conjugate with sodium borohydride resulting in an immunogenicconjugate comprising Streptococcus pneumoniae serotype 1 polysaccharidecovalently linked to a carrier protein.
 14. The method of claim 13,further comprising purifying the immunogenic conjugate.
 15. The methodof claim 13, wherein the carrier protein is CRM₁₉₇.
 16. A method formaking an activated Streptococcus pneumoniae serotype 1 polysaccharide,the method comprising: (a) reacting purified serotype 1 polysaccharidewith an alkaline pH buffer resulting in a partially de-O-acetylatedserotype 1 polysaccharide; (b) reacting the partially de-O-acetylatedserotype 1 polysaccharide with a mild acid resulting in a neutralized,partially de-O-acetylated serotype 1 polysaccharide; and (c) reactingthe neutralized, partially de-O-acetylated serotype 1 polysaccharidewith an oxidizing agent resulting in an activated serotype 1polysaccharide.
 17. The method of claim 16, further comprising purifyingthe activated serotype 1 polysaccharide following oxidation.
 18. Themethod of claim 16, wherein the alkaline pH buffer is abicarbonate/carbonate buffer.
 19. The method of claim 16, wherein themild acid is acetic acid.
 20. The method of claim 16, wherein theoxidizing agent is sodium periodate.
 21. A purified Streptococcuspneumoniae serotype 1 polysaccharide that is de-β-acetylated toapproximately 40 mole percent.
 22. The purified Streptococcus pneumoniaeserotype 1 polysaccharide of claim 21, wherein said polysaccharide isconjugated to a carrier protein.
 23. The purified Streptococcuspneumoniae serotype 1 polysaccharide of claim 21, wherein saidpolysaccharide is obtained by reacting purified Streptococcus pneumoniaeserotype 1 polysaccharide with an alkaline pH buffer.
 24. The purifiedStreptococcus pneumoniae serotype 1 polysaccharide of claim 23, whereinsaid polysaccharide is conjugated to a carrier protein.
 25. The purifiedStreptococcus pneumoniae serotype 1 polysaccharide of claim 23, whereinsaid alkaline pH buffer is a sodium bicarbonate/carbonate buffer at pH9.0.
 26. The purified Streptococcus pneumoniae serotype 1 polysaccharideof claim 25, wherein said buffer concentration is 30 mM.